The objectives of the proposed research are to understand the primary action of narcotic analgesics and the way in which these alter during the development of tolerance and physical dependence. Intracellular recordings will be made from neurons in the myenteric plexus of the guinea-pig ileum. Acute actions of opiates and opioid peptides will be studied on single membrane ion channels using intracellular voltage clamp (single electrode) combined with extracellular patch clamp recording. The effects of known concentrations of various opiate receptor subtype selective ligands will be applied. The role of intracellular and extracellular calcium ions will be assessed by applying calcium selectively to the inside of the plasma membrane using liposomes. Effects of opiates on transmitter release will be determined by recording synaptic potentials resulting from activity in single fibres. The longer term actions of opiates will be studied by repeating these experiments in neurons removed from guinea-pigs treated with morphine during several days. In addition, adult neurons will be maintained for several days or weeks as a primary explant culture, either with or without morphine. The results are expected to elucidate the primary mechanism of action of narcotic analgesics and opioid peptides on the membrane of excitable cells in both naive and drug dependent animals, and to provide insight into the way in which opiates exert their pharmacological effects and induce tolerance and dependence.